The diagnosis and antiviral therapy of HIV/AIDS is a major challenge in developing countries. Thus, the accurate determination of CD4+ T-cells is of major importance. Different trials have been started to develop an international external quality control for this immunological measurement. The main problem with implementing inter-laboratory tests in countries with resource-limited settings is the insufficient stability of fresh blood samples. For an international distribution, stabilized blood samples are required which can withstand shipment throughout the global, mainly tropical hemisphere. In order to solve this problem we have developed a new method of generating freeze-dried leukocytes which are suitable for long-term storage at elevated temperatures CD4+ T-cells are also known as helper T-cells and act as an co-ordinator of the immune response, unfortunately, CD4+ T-cells are also the main targets of HIV. HIV destroys infected CD4+ T-cells and leading to an overall weakening of the immune system.
The monitoring of CD4 T-cell counts has been well established as a surrogate marker for the therapy of HIV/AIDS (Phillips et al. 2008, Lancet 317:1443; Paintsil et al. 2008, Pdiatr. Infect Dis J; 27(7):629). The decision of when to switch the therapeutic regime from first to second line depends on the concentration of CD4+ T-cells (Ledergerber et al. 2004, Lancet: 364:51).
Lower numbers of circulating CD4+ T-cells indicates a weakening of the immune system and advancement in the progression of HIV disease. The CD4+ T-cell count can also be indicative of the success or failure of anti-retroviral therapy (ARV).
In countries with a high prevalence of HIV/AIDS and resource-limited settings, different affordable devices based on flow cytometry are available to determine the CD4+ count and CD4+ percentage of lymphocytes. Thus, the accuracy of measurements in regional testing facilities is a critical point involving several problems. To date, no international external quality assurance/quality control (QA/QC) programs for CD4 count and CD4 percentage in resource-limited settings exist. This is mainly due to the fact that cold chains or refrigeration during transportation and storage of control materials cannot be realized in many countries within the southern hemisphere.
Cytometric monitoring is being used for most of the 2 million patients currently receiving antiretroviral therapy in these countries. Commercially available fluid control materials like Cyto-Chex (Streck, USA) or Transfix (Cytomark, UK), based on stabilized blood cells, have a limited shelf life and are not stable enough for long transportation routes at elevated temperatures (Plate et al. 2009, Viral Immunol. 22(5):329; Truett et al. 2006, J Acquir Immune Defec Syndr, 41(2): 168). The effect of storage temperature has been investigated in several publications using different approaches to achieve blood preservation.
The correct determination of the CD4+ count and CD4+ percentage has significant implications for effective management of the individual antiretroviral HIV therapy (O'Gorman et al. 2008, Cytometry B Clin Cytom 74 Suppl 1:19). Therefore, it is essential that external QA/QC programs are implemented in countries with a high prevalence of HIV/AIDS. Several attempts have already been made to generate suitable control materials for flow cytometric CD4 analysis in regional facilities (Bergeron et al. 2009, Cytometry B Clin Cytom). Without a proper cooling chain, no durable cellular control material that contains leukocytes for flow cytometric CD4 monitoring is available to date. Commonly used preparations consist of chemically stabilized suspensions of whole blood with a maximum shelf life at ambient temperature (25-37° C.) of 2 weeks (Pattanapanyasat et al. 2005, J Med Res; 121(4):539). In practice, this time frame is often not sufficient for reliable usage in QA/QC programs. Currently used stable materials for internal or external quality controls are based on polystyrene or latex beads. Control beads and native blood samples are generally processed with different instrument settings. Concerning this matter, the usage of preserved human leukocytes forflow cytometric QC/QA has the advantage of being a full process control.
Until now, no adequate method has been found to preserve leukocytes with a durability of more than 2 weeks at 37° C. (Plate et al. 2009, Viral Immunol. 22(5):329), let alone for the conditions regarding transport times and ambient temperature prevailing in many countries within the southern hemisphere. Commercially available lyophilized lymphocytes or polystyrene and latex beads have the disadvantage of being almost artificial because they do not exhibit the same flow cytometric appearance as native blood, which is an important requirement for QA/QC programs.
Therefore, the problem of the invention to be solved was to provide a method of stabilizing leukocytes from human blood samples, wherein the stabilized leukocytes are comparable in function and activity with leukocytes freshly isolated from blood.